The objective of this project is to develop reliable and reproducible exposure assessment methodologies and protein expression-based methods aimed at the development of biological/ toxicological response endpoints that can serve potential biomarkers for well-characterized engineered nanomaterials (ENMs). While raw, unrefined, and hydrophobic carbon nanotubes tend to settle out of aqueous media/environs, water dispersible, functionalized carbon nanotubes (f-CNTs) will contaminate water resources. Large quantities of these derivatized forms of CNTs will be manufactured as they find diverse applications ranging from polymer composites to drug delivery. Accordingly, one of the major potential routes of human exposure is alimentary, occurring via consumption of water or products from plants and animals that have taken up the CNTs through food-chain dynamics. Reliable and reproducible methods to assess exposure and biological response/toxicological endpoints for well-characterized engineered nanomaterials (ENMs) must be developed. It is hypothesized that highly dispersible, water soluble carbon nanotubes alter protein expression in intestinal and liver cells as biological/toxicological endpoints of exposure and injurious effect in the gastrointestinal tract. These effects will be evident as intra- and/or extracellular protein differences, detectable by proteomic analysis and can be rendered as a panel of protein biomarker candidates. This has lead to the following aims: Specific Aim 1. f- CNTs will be synthesized, characterized, and analytical methodologies for exposure assessment developed. Composition, morphology and size of the functionalized CNTs will be assessed, as will residual impurities in an unrefined, comparator group. CNT-induced changes in cellular function and protein expression will be distinguished among physico-chemical characteristics of the individual CNTs. This will be done by element mapping analysis, X-ray energy dispersive spectrometry (EDS) in scanning electron microscopy (SEM), thermogravimetry analysis (TGA) and Raman Spectroscopy. Specific Aim 2. The effects of f-CNT exposure on colonic and hepatic epithelial cell protein expression profiles will be assessed to discover potential protein biomarkers of exposure/effect. Protein expression data will be obtained by two complementary methods: two- dimensional electrophoresis (2-DE) and label-free quantitative mass spectrometry (LFQMS). This will enable the analysis of relevant organelle, compartment, and pathway-specific responses to f-CNT exposure and identification of biological response/toxicological endpoints of ENM exposure. This project combines complementary expertise in nanotechnology and nanotoxicology using novel applications of emerging analytical techniques to study a relevant yet previously ignored target for water dispersible CNTs, the gut-liver axis. The results of this project will establish reproducible ENM characterization methodologies and lay the groundwork for the validation of individual biomarkers or biomarker panels that can reliably detect and predict biological response to select model ENMs, thereby promoting the safe use of these rapidly proliferating materials and protecting the public. PUBLIC HEALTH RELEVANCE: This project will develop reproducible Engineered Nanomaterial (ENM) characterization methodologies and generate protein biomarker candidates of biological/toxicological effect in cells of the digestive tract. These studies will lay the groundwork for the validation of individual biomarkers or biomarker panels that can reliably detect and predict biological response to well-characterized water dispersible ENMs, thereby promoting the safe use of these rapidly proliferating materials and protecting the public.